Simon Toze

PCR AND THE DETECTION OF MICROBIAL PATHOGENS IN WATER AND WASTEWATER

Microbial pathogens are one of the major health risks associated with water and wastewaters. Current methods for the detection of pathogenic viruses, bacteria, protozoa and helminths tend to be inaccurate, time consuming and expensive. As a result, indicator bacteria are commonly used to determine the relative risk of faecal contamination and the possible presence of pathogens in water and wastewaters. Indicator organisms, however, have several disadvantages that make them less than ideal for indicating the possible presence of microbial pathogens. Consequently methods to directly detect microbial pathogens in water and wastewaters are being investigated. Polymerase chain reaction (PCR) is one of the main alternative detection methods being trialed. PCR has been shown to be a rapid, highly sensitive and accurate method. It has already been used experimentally to detect pathogenic viruses, bacteria and protozoa in water and wastewaters. PCR, however, has a number of limitations. One such limitation is the generation of false positives through the detection of naked nucleic acids, non-viable microorganism, or through contamination in the laboratory. Other limitations include inhibition by environmental contaminants and difficulty in quantification. The potential of PCR for the direct detection of microbial pathogens in water and wastewater, as well as the current limitations are discussed below.

Human pathogens and their indicators in biosolids: A literature review

A growing beneficial reuse of biosolids in agriculture has led to concerns about potential contamination of water resources and the food chain. In order to comprehend the potential risks of transmission of diseases to the human population, an advanced quantitative risk assessment is essential. This requires good quantitative data which is currently limited due to the methodological limitations. Consequently, further development and standardization of methodologies for the detection, enumeration and viability assessment of pathogens in biosolids is required. There is a paucity of information on the numbers and survival of enteric virus and protozoan pathogens of concern in biosolids. There is a growing urgency for the identification of more reliable alternative indicators, both index and model microorganisms, which could be used for potential public health risk assessment. In this review, we have summarized reported literature on the numbers and fate of enteric pathogens and indicators in biosolids. The advantages and limitations of the use of conventional and alternative index and model microorganisms for the prediction of pathogen presence in biosolids are also discussed.

Influence of groundwater characteristics on the survival of enteric viruses

Aims: This study was undertaken to further understand the processes affecting the persistence of enteric viruses in groundwater.

Sewage pollution in urban stormwater runoff as evident from the widespread presence of multiple microbial and chemical source tracking markers.

The concurrence of human sewage contamination in urban stormwater runoff (n=23) from six urban catchments across Australia was assessed by using both microbial source tracking (MST) and chemical source tracking (CST) markers. Out of 23 stormwater samples human adenovirus (HAv), human polyomavirus (HPv) and the sewage-associated markers; Methanobrevibacter smithii nifH and Bacteroides HF183 were detected in 91%, 56%, 43% and 96% of samples, respectively. Similarly, CST markers paracetamol (87%), salicylic acid (78%) acesulfame (96%) and caffeine (91%) were frequently detected. Twenty one samples (91%) were positive for six to eight sewage related MST and CST markers and remaining two samples were positive for five and four markers, respectively. A very good consensus (>91%) observed between the concurrence of the HF183, HAv, acesulfame and caffeine suggests good predictability of the presence of HAv in samples positive for one of the three markers. High prevalence of HAv (91%) also suggests that other enteric viruses may also be present in the stormwater samples which may pose significant health risks. This study underscores the benefits of employing a set of MST and CST markers which could include monitoring for HF183, adenovirus, caffeine and paracetamol to accurately detect human sewage contamination along with credible information on the presence of human enteric viruses, which could be used for more reliable public health risk assessments. Based on the results obtained in this study, it is recommended that some degree of treatment of captured stormwater would be required if it were to be used for non-potable purposes.

The Biogeography and Phylogeny of Unicellular Cyanobacterial Symbionts in Sponges from Australia and the Mediterranean

The distribution, host associations, and phylogenetic relationships of the unicellular cyanobacterial symbionts of selected marine sponges were investigated with direct 16s rDNA sequencing. The results indicate that the symbionts of the marine sponges Aplysina aerophoba, Ircinia variabilis, and Petrosia ficiformis from the Mediterranean, four Chondrilla species from Australia and the Mediterranean, and Haliclona sp. from Australia support a diversity of symbionts comprising at least four closely related species of Synechococcus. These include the symbionts presently described as Aphanocapsa feldmannii from P. ficiformis and Chondrilla nucula. A fifth symbiont from Cymbastela marshae in Australia is an undescribed symbiont of sponges, related to Oscillatoria rosea. One symbiont, Candidatus Synechococcus spongiarum, was found in diverse sponge genera in the Mediterranean Sea and the Indian, Pacific, and Southern oceans, whereas others were apparently more restricted in host association and distribution. These results are discussed in terms of the biodiversity and biogeographic distributions of cyanobacterial symbionts.

Use of static Quantitative Microbial Risk Assessment to determine pathogen risks in an unconfined carbonate aquifer used for Managed Aquifer Recharge.

Managed Aquifer Recharge (MAR) is becoming a mechanism used for recycling treated wastewater and captured urban stormwater and is being used as a treatment barrier to remove contaminants such as pathogens from the recharged water. There is still a need, however, to demonstrate the effectiveness of MAR to reduce any residual risk of pathogens in the recovered water. A MAR research site recharging secondary treated wastewater in an unconfined carbonate aquifer was used in conjunction with a static Quantitative Microbial Risk Assessment (QMRA) to assess the microbial pathogen risk in the recovered water following infiltration and aquifer passage. The research involved undertaking a detailed hydrogeological assessment of the aquifer at the MAR site and determining the decay rates of reference pathogens from an in-situ decay study. These variables along with literature data were then used in the static QMRA which demonstrated that the recovered water at this site did not meet the Australian Guidelines for recycled water when used for differing private green space irrigation scenarios. The results also confirmed the importance of obtaining local hydrogeological data as local heterogeneity can influence of residence time in the aquifer which, in turn, influences the outcomes. The research demonstrated that a static QMRA can be used to determine the residual risk from pathogens in recovered water and showed that it can be a valuable tool in the preliminary design and operation of MAR systems and the incorporation of complementary engineered treatment processes to ensure that there is acceptable health risk from the recovered water.

Prevalence of clinically relevant antibiotic resistance genes in surface water samples collected from Germany and Australia

The prevalence and proliferation of antibiotic resistant bacteria is profoundly important to human health, but the extent to which aquatic environments contribute toward the dissemination of antibiotic resistant genes (ARGs) is poorly understood. The prevalence of 24 ARGs active against eight antibiotic classes (β-lactams, aminoglycosides, glycopeptides, chloramphenicols, tetracycline, macrolides, trimethoprim, and sulfonamides) was evaluated in surface water samples collected from Germany and Australia with culture independent methods. The ARGs most frequently detected both in Germany and Australia were sulI, sulII (77-100%), and dfrA1 (43-55%) which code for resistance to sulfonamide and trimethoprim. Macrolides resistance gene ermB was relatively more prevalent in the surface water from Germany (68%) than Australia (18%). In contrast, the chloramphenicol resistance gene catII was more frequently detected in Australia (64%) than Germany (9%). Similarly, β-lactams resistance gene ampC was more prevalent in the samples from Australia (36%) than Germany (19%). This study highlights wide distribution of ARGs for sulfonamide, trimethoprim, macroline, β-lactams and chloramphenicol in the aquatic ecosystems. Aquatic ecosystems can therefore be reservoirs of ARGs genes which could potentially be transferred from commensal microorganisms to human pathogens.

Bioanalytical tools for the evaluation of organic micropollutants during sewage treatment, water recycling and drinking water generation.

A bioanalytical test battery was used for monitoring organic micropollutants across an indirect potable reuse scheme testing sites across the complete water cycle from sewage to drinking water to assess the efficacy of different treatment barriers. The indirect potable reuse scheme consists of seven treatment barriers: (1) source control, (2) wastewater treatment plant, (3) microfiltration, (4) reverse osmosis, (5) advanced oxidation, (6) natural environment in a reservoir and (7) drinking water treatment plant. Bioanalytical results provide complementary information to chemical analysis on the sum of micropollutants acting together in mixtures. Six endpoints targeting the groups of chemicals with modes of toxic action of particular relevance for human and environmental health were included in the evaluation: genotoxicity, estrogenicity (endocrine disruption), neurotoxicity, phytotoxicity, dioxin-like activity and non-specific cell toxicity. The toxicity of water samples was expressed as toxic equivalent concentrations (TEQ), a measure that translates the effect of the mixtures of unknown and potentially unidentified chemicals in a water sample to the effect that a known reference compound would cause. For each bioassay a different representative reference compound was selected. In this study, the TEQ concept was applied for the first time to the umuC test indicative of genotoxicity using 4-nitroquinoline as the reference compound for direct genotoxicity and benzo[a]pyrene for genotoxicity after metabolic activation. The TEQ were observed to decrease across the seven treatment barriers in all six selected bioassays. Each bioassay showed a differentiated picture representative for a different group of chemicals and their mixture effect. The TEQ of the samples across the seven barriers were in the same order of magnitude as seen during previous individual studies in wastewater and advanced water treatment plants and reservoirs. For the first time a benchmarking was performed that allows direct comparison of different treatment technologies and covers several orders of magnitude of TEQ from highly contaminated sewage to drinking water with TEQ close or below the limit of detection. Detection limits of the bioassays were decreased in comparison to earlier studies by optimizing sample preparation and test protocols, and were comparable to or lower than the quantification limits of the routine chemical analysis, which allowed monitoring of the presence and removal of micropollutants post Barrier 2 and in drinking water. The results obtained by bioanalytical tools were reproducible, robust and consistent with previous studies assessing the effectiveness of the wastewater and advanced water treatment plants. The results of this study indicate that bioanalytical results expressed as TEQ are useful to assess removal efficiency of micropollutants throughout all treatment steps of water recycling.

Escherichia coli survival in groundwater and effluent measured using a combination of propidium iodide and the green fluorescent protein

Aims:  The aim of this study was to deterimine the survival of an enteric bacterium in anaerobic groundwater and effluent microcosms using the green fluorescent protein (GFP) marker gene in combination with the viability indicator propidium iodide (PI).

Prevalence of human pathogens and indicators in stormwater runoff in Brisbane, Australia

Elevated numbers of enteric pathogens in the receiving waters following a storm event can be a serious public health concern. The purpose of this study was to conduct a preliminary investigation into the presence of human pathogens of concern in urban stormwater runoff. The involvement of a human sewage as a potential source of contamination was also investigated by using microbial source tracking methods. Water samples (20 L) were collected after storm events and during the dry weather from six sites in Brisbane, Australia. Collected samples were analyzed for fecal indicator bacteria (FIB), and then concentrated using hollow fiber ultrafiltration followed by molecular detection of selected enteric pathogens. The levels of FIB were found to frequently exceed the upper limit of Australian guidelines for managing risks in recreational water, during the dry periods and by further several orders of magnitude in the stormwater runoff. Enterococcus spp. numbers as high as 3×10(4) 100 mL(-1) were detected in the stormwater runoff at the Fitzgibbon site. Human adenovirus and polyomavirus were frequently detected from all six sampling sites during wet and dry weather conditions suggesting their wide spread presence in the urban aquatic environments. Campylobacter jejuni, Campylobacter coli and Salmonella enterica were also detected during both dry and wet weather conditions. Presence of human-specific HF183 Bacteroides marker in most of the samples tested suggests ubiquitous sewage contamination in the urban environment. Since stormwater runoff routinely contains high numbers of FIB and other enteric pathogens, some degree of treatment of captured stormwater would be required if it were to be used for non-potable purposes.